Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Interaction of Glufosinate and <i>Colletotrichum truncatum</i>on Ammonia Levels and Glutamine Synthetase Activity in Hemp Sesbania

The use of microbes and microbial products as bioherbicides has been studied for several decades, and combinations of bioherbicides and herbicides have been examined to discover possible synergistic interactions to improve weed control efficacy. Bioassays were conducted to assess possible interactions of the herbicide glufosinate [2-amino-4-(hydroxymethylphosphinyl) butanoic acid] and Colletotrichum truncatum (CT), a fungal bioherbicide to control hemp sesbania (Sesbania exaltata)]. Glufosinate acts as a glutamine synthetase (GS) inhibitor that causes elevated ammonia levels, but the mode of action of CT is unknown. GS has also been implicated in plant defense in certain plant-pathogen interactions. The effects of spray applications of glufosinate (1.0 mM) orbioherbicide (8.0 × 104 conidia ml-1), applied alone or in combination were monitored (88 h time-course) on seedling growth, GS activity and ammonia levels in hypocotyl tissues under controlled environmental conditions. Growth (elongation and fresh weight) and extractable GS activity were inhibited in tissues by glufosinate and glufosinate plus CT treatments as early as 16 h, but CT treatment did not cause substantial growth reduction or GS inhibition until after ~40 h. Generally, ammonia levels in hemp sesbania tissues under these various treatments were inversely correlated with GS activity. Localization of hemp sesbania GS activity on electrophoretic gels indicated a lack of activity after 30 h in glufosinate and glufosinate plus CT-treated tissue. Untreated control tissues contained much lower ammonia levels at 24, 64, and 88 h after treatment than treatments with CT, glufosinate or their combination. CT alone caused elevated ammonia levels only after 64 - 88 h. Glufosinate incorporated in agar at 0.25 mM to 2.0 mM, caused a 10% - 45% reduction of CT colony radial growth, compared to fungal growth on agar without glufosinate, and the herbicide also inhibited sporulation of CT. Although no synergistic interactions were found in the combinations of CT and glufosinate at the concentrations used, further insight on the biochemical action of CT and its interactions with this herbicide on hemp sesbania was achieved.

Efficacy of 2,4-D, Dicamba, Glufosinate and Glyphosate Combinations on Selected Broadleaf Weed Heights

Palmer amaranth, sicklepod and pitted morningglory are the three most common and troublesome weeds in soybean in South Carolina. They exhibit very aggressive growth capabilities and if left uncontrolled in fields will cause significant reductions in soybean yields. Dicamba and 2,4-D herbicides are currently having a resurgence in usage due to the recent commercialization of soybean trait technologies with tolerance to these herbicides. Dicamba and 2,4-D when tank mixed with glufosinate and glyphosate may offer additional weed control to resistant weeds through the process of herbicide synergism. Greenhouse experiments were conducted in 2013 at Edisto Research and Education Center near Blackville, SC to evaluate the efficacy of glyphosate, glufosinate, dicamba and 2,4-D treatments alone and in combination on Palmer amaranth, sicklepod, and pitted morningglory at selected heights. Results suggested that glufosinate alone provided the overall best control for all 3 weed species. Glyphosate alone provided the lowest control of all 3 species at all heights. Synergism or improved sicklepod control was observed when glufosinate was tank mixed with dicamba. However, as sicklepod increased in height, glufosinate + 2,4-D or dicamba combination offered the best control compared to glufosinate alone (90% versus 86% in 20 cm plants and 87% versus 85% in 30 cm plant). In the 5 cm Palmer amaranth, decreased control was observed when glyphosate or glufosinate was tank mixed with 2,4-D. These experiments showed that glufosinate alone and/or in combination with 2,4-D or dicamba was the overall best treatment on the three broadleaf weed species.

Separation and Purification of Glufosinate Through Combination of an Electrodialysis Membrane and a Macroporous Adsorption Resin

Glufosinate is a nonselective organophosphorus herbicide with low toxicity and high efficiency that is widely used in forestry, agriculture and other industries. In the process of manufacturing glufosinate, large amounts of ammonium chloride and coloured organic impurities are generated. Because of its high solubility in water, separation of glufosinate from inorganic salts is extremely difficult. Hence, a co-separation method combining an electrodialysis membrane and a macroporous adsorption resin was developed to obtain glufosinate with higlier purity. For the electrodialysis process, a glufosinate reaction solution was placed in a dilute chamber and desalinated. The concentration of inorganic salts in the resultant glufosinate aqueous solution was only 0.99 g/L under the optimal conditions of an operating voltage and a volume ratio of 9 V and 1:1, respectively. For the macroporous resin adsorption/desorption process, the sample solution treated by electrodialysis was pumped into the resin-filled column, which was eluted to obtain the eluent when the adsorption reached equilibrium. Consequently, nearly all the coloured organic impurities were removed under the optimal conditions, in which the resin type, pH value, flow rate, glufosinate concentration, temperature, ratio of ethanol and volume of eluent were LX-300C,3,0.5 mL·cm^2·min^-1,20 mg/mL,25℃, 50% and 400 mL, respectively. After the electrodialysis and adsorption/desorption process, the purity of the glufosinate was increased to 95.14%, and its recovery rate was as high as 98%. The advantages of this process included its ease of operation, environmental friendliness and low cost, which provided strong potential for its use in industrial applications.

不同施氮水平下GS抑制剂对小麦灌浆期碳氮代谢的影响

为了解小麦叶片和籽粒内碳代谢和氮代谢之间的关联性,采用盆栽试验,研究了谷氨酰胺合成酶(GS)抑制剂(Glufosinate)对不同施氮水平下两个小麦基因型灌浆期氮、碳同化特性的影响及其相互关系。结果表明,叶片GS被抑制后,高蛋白类型品种豫麦47籽粒内氮含量显著升高、叶片内可溶性糖含量上升;而低蛋白类型豫麦50则表现出相反的趋势。穗GS被抑制后,高蛋白类型品种豫麦47叶片和籽粒内氮含量均显著下降、叶片和籽粒内的可溶性糖含量也显著下降;而低蛋白类型豫麦50则表现叶片和籽粒内氮含量均显著下降,但可溶性糖含量显著上升。说明高蛋白品种豫麦47籽粒和叶片内氮、碳同化相对独立,而低蛋白品种豫麦50籽粒和叶片内氮、碳同化过程关联紧密。这一结果还说明高蛋白品种籽粒花后氮同化可以直接利用从根部或茎秆而来的氮素,而低蛋白品种则必须经过叶片。因此,在小麦生产上,不同类型品种必须采取不同的氮肥运筹方案,对于低蛋白类型品种,氮肥可适当前移,而对于高蛋白类型品种,则宜采用前促后补的策略。

Glyphosate-Resistant Common Ragweed Control in Corn with Postemergence Herbicides

Four field trials were conducted on a farm infested with glyphosate-resistant (GR) common ragweed during 2016 and 2017 to evaluate various postemergence (POST) herbicides for the control of GR common ragweed in GR corn. Dicamba at 600 g·a.i.·ha-1, dicamba/diflufenzopyr at 200 g·a.i.·ha-1, dicamba/atrazine at 1500 g·a.i.·ha-1, topramezone + atrazine at 12.5 + 500 g·a.i.·ha-1, bromoxynil + atrazine at 280 + 1500 g·a.i.·ha-1, glufosinate at 500 g·a.i.·ha-1 and 2,4-D ester at 560 g·a.i.·ha-1 provided 58% to 85% control at 4 WAA and 49% to 88% control at 8 WAA. Other herbicides evaluated controlled GR common ragweed 9% to 41%. Common ragweed density was reduced 97%, 95%, 95% and 87% and shoot dry weight was reduced 93%, 95%, 94% and 90% with bromoxynil + atrazine, dicamba, glufosinate and topramezone + atrazine applied POST in GR corn, respectively. Results show that dicamba, bromoxynil + atrazine, topramezone + atrazine and glufosinate applied POST are the most efficacious herbicides among the herbicides evaluated for the control of GR common ragweed in GR corn.

Efficacy of Selected Herbicide Programs in 2,4-D Tolerant Cotton (<i>Gossypium hirsutum</i>L.)

The use of transgenic crops has grown significantly over the past couple of decades. Many agronomic crops produced today are tolerant to glyphosate. Glyphosate-tolerant crops were commercially introduced in 1996, and, about nine years later, glyphosate-resistant Palmer amaranth was confirmed in Georgia. Glyphosate-resistant weeds arose from reliance on postemergence only glyphosate programs to control weeds in crops. New transgenic traits for glufosinate and 2,4-D choline have been developed, and evaluations of stacked traits and concurrent use of multiple herbicides have provided additional tools in the management of glyphosate-resistant weeds. Field experiments were conducted in 2012 and 2013 at the Edisto Research and Education Center near Blackville, SC, USA to determine the efficacy of 2,4-D-based herbicide programs in transgenic cotton tolerant to 2,4-D choline, glyphosate, and glufosinate. The treatments provided good to excellent Palmer amaranth and pitted morningglory control in 2012 and 2013. Seed cotton yields across treatments ranged from 0 to 2057 kg ha-1. This new trait technology package in cotton permits in-season postemergence use of 2,4-D choline, a herbicide mode of action not previously used postemergence in cotton, which can control resistant weeds, including Palmer amaranth if applied at the proper growth stage.

福建大豆主栽品种的组织培养研究

利用福建大豆主栽品种的子叶节为外植体,进行组织培养再生研究,探讨不同品种、不同激素和除草剂不同浓度的诱导效果。结果表明不同大豆品种诱导分化能力差别较大,供试品种中“莆豆8008”的诱导能力较强,丛生芽诱导率和平均丛生芽数分别为94.2%和2.69。不同激素种类和浓度对大豆子叶节丛生芽诱导,表明6-BA较适合于丛生芽的诱导,适宜浓度为1.67mg/L。大豆子叶节不定芽分化和伸长的除草剂Glufosinate梯度实验表明除草剂Glufosinate5mg/L可作为不定芽分化和伸长的选择压力,生根培养基上不宜加除草剂Glufosinate。